EP0101979B1 - Diagnostic device and its use - Google Patents

Abstract

A diagnostic device for the detection of an increased concentration of dehydrogenases and/or oxidases in the fluids of humans, animals or plants, which consists of a carrier which bonds a redox dyestuff, and a substance mixture, adjusted to a pH value in the acid range, of the substrate corresponding to the particular dehydrogenase, a hydrogen donor compound and at least one redox dyestuff is described. The device has the peculiarity that the carrier contains polar groups and the substance mixture for detecting exclusively pathologically increased concentrations of the particular dehydrogenase and/or oxidase is adjusted to a pH value of below 5.0. The device is suitable for diagnosing malignant growths in very different organs, for example in the female genital area.

Description

The invention relates to a diagnostic device for detecting an increased concentration of dehydrogenases and / or oxidases and the use of the device.

For each cytosolic dehydrogenase that changes into extracellular fluids in the event of a pathological change in humans, animals or plants, the pathological threshold value can be empirically determined in these fluids. When checking for a pathological change, the physico-chemical parameters of the diagnostic system should be set so that the pathological threshold value of the dehydrogenase or oxidase activity under the diagnostic conditions with the point of transition of an indicator, e.g. of a redox dye. In the case of pathological changes in the properties of cell membranes, it is to be expected that various pyridine-dependent dehydrogenases, such as lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase, glutamate dehydrogenase, glucose dehydrogenase and glucose , and / or flavin-dependent dehydrogenases and / or flavin-dependent oxidase, such as succinate dehydrogenase, glycerol-3-phosphate dehydrogenase, glucose oxidase, pass into the serum and into other extracellular liquids and one for each dehydrogenase or oxidase the diagnosis of pathological threshold is found.

• a. Es wird das Substrat der jeweiligen Dehydrogenase eingesetzt, z. B. Lactat für LDH.
• b. Die Mengen des vorgelegten Substrats und des gleichzeitig eingesetzten Pyridinnucleotids werden eingestellt.
• c. Der pH-Wert und damit die Lage des Reaktionsgleichgewichts wird eingestellt, gegebenenfalls mittels eines Puffersystems.
• d. Es wird ein Redox-Farbstoff (z.B. Leukofarbstoff, der von farblos nach farbig umschlägt) gewählt, dessen Redox-Potential unter den Diagnosebedingungen über dem Redox-Potential des Pyridinnucelotid-Systems liegt.
• e. Es wird ein Elektronenüberträger gewählt, dessen Redox-Potential zwischen dem des Pyridinnucleotid-Systems und dem des gewählten Redox-Farbstoffs liegt.
• f. Solange das Gleichgewicht der Dehydrogenase-Reaktion noch nicht eingestellt ist, ergibt eine längere Umsetzungszeit eine höhere Konzentration des reduzierten Pyridinnucleotids. Dementsprechend kann die Umschlagsschwelle des Redox-Farbstoffs und damit die Nachweisempfindlichkeit des Diagnosesystems über die Umsetzungsdauer eingestellt werden.
• g. Es können unterschiedliche Träger für das Diagnosesystem gewählt werden.

As a pathological change here comes z. B. consider the formation of malignancies. To adapt the transition point of a redox dye to the respective pathological threshold value, the physico-chemical parameters of the diagnostic system can, for. B. can be set as follows:

• a. The substrate of the respective dehydrogenase is used, e.g. B. Lactate for LDH.
• b. The amounts of the substrate and the pyridine nucleotide used at the same time are adjusted.
• c. The pH and thus the position of the reaction equilibrium is adjusted, if necessary using a buffer system.
• d. A redox dye (for example leuco dye which changes from colorless to colored) is selected whose redox potential is above the redox potential of the pyridine nucelotide system under the diagnostic conditions.
• e. An electron carrier is selected whose redox potential lies between that of the pyridine nucleotide system and that of the selected redox dye.
• f. As long as the equilibrium of the dehydrogenase reaction has not yet been established, a longer reaction time results in a higher concentration of the reduced pyridine nucleotide. Accordingly, the turnover threshold of the redox dye and thus the detection sensitivity of the diagnostic system can be set over the implementation period.
• G. Different carriers can be selected for the diagnostic system.

From DE-PS 2 443 741 a method for the joint determination of isozymes 4 and 5 of the LDH is known, in which a mixture of a lactate and nicotinamide adenine dinucleotide (NAD) is brought into contact with the body fluid to be examined. If LDH is present in an increased amount, it catalyzes the conversion of the lactate with the dinucleotide to the corresponding pyruvate and the reduced nicotinamide adenine dinucleotide (NADH) according to the following equation:

LDH occurs in human cells in the form of isoenzymes 1, 2, 3, 4 and 5. For certain specific pathological changes, elevated LDH values occur, e.g. B. Malignancies in the female genital area lead to an increase in isoenzymes 4 and 5.

• Nitroblauformazan [4,4'-(3,3'-Dimethoxy-biphe- nylenyl)-bis-2N-(2N'-nitrophenyl-C-phenyl- formazan)]
• übergeht und so die enzymatische Aktivität und Anwesenheit der LDH bzw. ihrer Isoenzyme sichtbar werden lässt.

According to the publication, the reaction between the lactate and the NAD must be carried out at a pH of 6 to 6.5. For this purpose, the body fluid and / or the lactate and the NAD-containing test reagent are adjusted to this pH range, if necessary using a buffer system. In cases where a body fluid, such as vaginal secretions, has a lower pH, e.g. B. has a pH of 4, the test reagent can be adjusted to a pH; which exceeds the value 6.5 so that the desired pH value in the range from 6 to 6.5 results in the test reaction. The conversion that runs to the right in the above formula equation is made visible by a subsequent reaction. For this purpose, the test reagent is mixed with nitroblue tetrazolium chloride (NBT) and optionally phenazine methosulfate (N-methylphenazonium methyl sulfate, 5-methylphenazonium methyl sulfate, PMS) as a hydrogen or electron carrier. The NADH formed according to the equation is oxidized by the NBT to NAD in the presence of the hydrogen or electron transfer, the NBT in the blue dye

• Nitroblauformazan [4,4 '- (3,3'-Dimethoxy-biphenylenyl) -bis-2N- (2N'-nitrophenyl-C-phenylformazan)]
• passes and so the enzymatic activity and presence of the LDH or their isoenzymes become visible.

The known method has been applied to the examination of the vaginal secretions of women. The duration of the reaction between this secretion and the diagnostic reagent was preferably 15 to 30 minutes. Pathological changes in the female genital tract, e.g. B. Carcinoma colli uteri, Carcinoma corporis and Carcinoma found in situ.

When checking for z. B. women on genital carcinoma by other known methods, e.g. B. cytology and colposcopy, false-negative diagnoses for collum and cervical carcinomas result in the order of magnitude of an average of 30% of the findings. This does not include any other female genital carcinomas than those listed above. There is therefore a great need to reduce this error rate.

GB-PS 893 301 describes a colorimetric test for serum enzymes. For the determination of lactate dehydrogenase in biological liquids, a mixture is specified there which contains a salt of lactic acid, diphosphopyridine nucleotide (nicotinamide adenine dinucleotide), diaphorase and 2,6-dichloroindophenol. The pH of the mixture should be in the range of 5 to 9.

However, according to this document, a pH below 7.0 was obviously not seriously considered. This is because the range given as preferred is pH 7 to 8, and a special embodiment is carried out at a pH of 7.4. Another passage states that the test is carried out in a solution carefully buffered with a pH 7 buffer. Finally, it has been shown that this test cannot actually be carried out at a pH of 6. The publication does not mention the use of tetrazolium salts in connection with the determination of LDH.

Otherwise, this known test is generally intended for the determination of serum enzymes, but not for the detection of exclusively pathologically elevated concentrations of dehydrogenases. Accordingly, the known test should respond even at very low enzyme concentrations, so that it is not possible to differentiate normal and pathologically increased dehydrogenase or oxidase values.

The invention has for its object to provide a diagnostic device for detecting a pathologically increased concentration of dehydrogenases in liquids or secretions of humans, animals or plants. The device is intended to ensure high diagnostic accuracy and is particularly suitable for the detection of malignancies. In addition, the diagnosis should be carried out as quickly as possible.

This object is achieved according to the invention by a diagnostic device which consists of a carrier which binds a redox dye and a mixture of substances adjusted to a pH in the acidic range from the substrate corresponding to the particular dehydrogenase, a hydrogen-transferring compound and at least one redox Dye exists. The device is characterized in that a carrier consisting of cellulose fibers and having polar groups is used and the mixture of substances is adjusted to a pH value of below 5.0 for the detection of exclusively pathologically elevated concentrations of the respective dehydrogenase and / or oxidase.

The diagnostic device is particularly well suited for the detection of pathological changes which are accompanied by an increase in the concentration of dehydrogenases in extracellular fluids. Such changes in concentration also occur in the early stages of malignancies, in which the prospect of successful therapy is particularly great. The diagnostic device gives an indication of malignancies on very different organs. Among other things, carcinomas on typical female organs, such as in the female genital area, can be detected. Infections by bacteria, parasites and fungi can also be detected using the device.

A particular advantage of the diagnostic device is its high predictive value of the diagnoses. For example, in series tests on women with histologically confirmed carcinomas in the genital area for stages I to IV, it was found that only less than 10% of all diagnoses are false negative.

In connection with this dye formation have special investigations in which, for. B. Tissue sections have also been included, show that the dye surprisingly remains on the support material during and after its formation and therefore does not trigger any harmful secondary reactions in and on the body of the examined patient.

These advantages make the diagnostic device particularly valuable for preventive medical examinations which are desirable in terms of health policy and in which not only the greatest possible degree of diagnostic certainty, but also a high degree of certainty against harmful secondary reactions of the diagnostic method must be given with a method that is as simple as possible.

The outstanding properties of the diagnostic device are due to the special combination of a carrier with polar groups and a low pH of the substance mixture on the carrier. It is surprising that this makes differential diagnosis possible, which records the pathological cases with high accuracy.

The great importance of the carrier for the function of the diagnostic device shows z. B. in that, without using the carrier, the color reaction of the substance mixture with a liquid containing a pathologically increased dehydrogenase concentration does not take place in the low pH range mentioned.

The liquid used for diagnosis can come from very different areas in humans, animals or plants. For example, the vaginal secretion can be used to test for malignant neoplasms in the area of the genital organs of women. The diagnostic device is also, for. B. on blood serum, stomach and Intestinal juice, bile, bronchial, prostate and urethral secretions and sputum can be used.

If the dehydrogenase to be detected is LDH, sodium lactate is preferably used. However, other corresponding connections, e.g. Potassium lactate or calcium lactate can be used.

The hydrogen-transferring compound is a substance that reduces the redox dye, e.g. a tetrazolium salt. Examples of such hydrogen-transferring compounds are NAD, nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). However, it is also possible to use other compounds familiar to the person skilled in the art which, for. B. in the conversion of lactate to pyruvate, instead of the NAD, can be converted into a reduced form corresponding to the NADH.

In order to visualize a dehydrogenase concentration in a liquid to be examined which is above the pathological threshold value, at least one redox indicator, preferably a redox dye, is contained in the substance mixture on the carrier.

• TNBT
• MTT
• INT
• BT
• TV
• TT
• NT
• Trinitro-TT
• Dinitro-TT
• Tetranitro-NT
• Tetranitro-BT
• Nitro-BT-methyl (Methoxygruppe substituiert durch Methylgruppe)
• Dinitro-TV
• 3-(4-Biphenyl)-2,5-di-p-nitrophenyl-tetrazoliumchlorid
• Nitro-NT
• Tetranitro-BT_I/2 (symmetrisch geteiltes Tetranitro-BT)
• Nitro-BT
• 3-(3,3'-Dimethoxy-4-biphenylylen)-2-p-nitrophenyl-5-phenyltetrazoliumchlorid
• Nitro-TV
• Nitro-TT
• 3-p-Jodophenyl-5-nitrophenyl-2-phenyl-tetrazoliumchlorid
• 5,5'-Dinitro-NT
• 5,5'-Dinitro-BT
• 5-Nitro-TV
• 5-Nitro-TT
• 2-(3-Methoxy-4-phenyJ)-5-p-nitrophenyl-3-phenyltetrazoliumchIorid Dabei bedeuten:
• NBT = 3,3'-(3,3'-Dimethoxy-4,4'-biphenylen)-bis-(2-p-nitrophenyl-5-phenyl-2H-tetrazoliumchlorid)
• TNBT = 3,3'-(3,3'-Dimethoxy-4,4'-bisphe-nylen)-bis-(2,5-p-nitrophenyl-2H-tetra-zolium- chlorid)
• MTT = 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromid
• INT = 2-p-Jodphenyl-3-p-nitrophenyl-5-phenyl-2H-tetrazoliumchlorid
• BT = 3,3'-(3,3'-Dimethoxy-4,4'-biphe- nyien)-bis-(2,5-diphenyi-2H-tetrazoliumchlorid)
• TV = 3a-Naphthyl-2,5-diphenyl-2H-tetrazoliumchlorid
• TT = 2,3,5-Triphenyl-2H-tetrazoliumchlorid NT = 3,3'-(4,4'-biphenylen)-bis-(2,5-diphenyl-2H-tetrazoliumchlorid
• ABTS = 2,2'-Azino-bis[3,3'-äthyl-benzthiazolin-6,6'-sulfonsäure]

As a dye component such. B. tetrazolium salts, benzidine dyes, indophenols, such as 2,6-dichloroindophenol or dibromoindophenol, or ABTS. Corresponding special tetrazolium salts are given below: NBT

• TNBT
• MTT
• INT
• BT
• TV
• TT
• NT
• Trinitro-TT
• Dinitro-TT
• Tetranitro-NT
• Tetranitro-BT
• Nitro-BT-methyl (methoxy group substituted by methyl group)
• Dinitro TV
• 3- (4-biphenyl) -2,5-di-p-nitrophenyl-tetrazolium chloride
• Nitro-NT
• Tetranitro-BT _{I / 2} (symmetrically split Tetranitro-BT)
• Nitro BT
• 3- (3,3'-Dimethoxy-4-biphenylylene) -2-p-nitrophenyl-5-phenyltetrazolium chloride
• Nitro TV
• Nitro-TT
• 3-p-iodophenyl-5-nitrophenyl-2-phenyl-tetrazolium chloride
• 5,5'-dinitro-NT
• 5,5'-dinitro-BT
• 5-nitro TV
• 5-nitro-TT
• 2- (3-methoxy-4-phenyJ) -5-p-nitrophenyl-3-phenyltetrazolium chloride where:
• NBT = 3,3 '- (3,3'-dimethoxy-4,4'-biphenylene) -bis- (2-p-nitrophenyl-5-phenyl-2H-tetrazolium chloride)
• TNBT = 3,3 '- (3,3'-dimethoxy-4,4'-bisphenylene) bis (2,5-p-nitrophenyl-2H-tetrazolium chloride)
• MTT = 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide
• INT = 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-2H-tetrazolium chloride
• BT = 3,3 '- (3,3'-dimethoxy-4,4'-biphenyien) -bis- (2,5-diphenyi-2H-tetrazolium chloride)
• TV = 3a-naphthyl-2,5-diphenyl-2H-tetrazolium chloride
• TT = 2,3,5-triphenyl-2H-tetrazolium chloride NT = 3,3 '- (4,4'-biphenylene) bis- (2,5-diphenyl-2H-tetrazolium chloride
• ABTS = 2,2'-azino-bis [3,3'-ethyl-benzthiazoline-6,6'-sulfonic acid]

With the above tetrazolium salts, the color change of the diagnostic device can be adjusted particularly well to the pathological threshold value of the dehydrogenases to be detected. Therefore, tetrazolium salts, especially NBT, are preferred as redox dyes.

The mixture of substances on the carrier of the diagnostic device has a pH of below 5.0, preferably 3.0 to below 5.0, in particular 4.5. It is surprising that the reaction that occurs when using the device e.g. is catalyzed by the LDH in such a low pH range because, as can be seen from the formula equation above, hydrogen ions are released during the conversion of the lactate to the pyruvate. One would therefore have expected that if the hydrogen ion concentration was increased further beyond a pH of 6.0, as is known from DE-PS 2 443 741, the reaction mentioned would proceed even more slowly or no longer, or that in the equation shown in the equation is shifted to the left. Contrary to these expectations, however, the reaction proceeds in the desired manner without any apparent slowdown, and, as mentioned, the diagnostic accuracy of the device according to the invention is also improved compared to known methods.

The setting of the desired pH of the mixture can be carried out in a conventional manner, e.g. B. by adding hydrochloric acid and / or phosphoric acid.

According to a preferred embodiment of the diagnostic device, the mixture of substances contains a buffer system for setting and / or maintaining the pH. As a result, the reaction medium is kept in a favorable pH range when the diagnostic device is used, even if the examined liquid has a different pH value.

The buffer system is preferably in the form of triethanoiamine hydrochloride or in the form of a non-equivalent mixture of triethanol amine and hydrogen chloride. The hydrogen chloride is used, for example, in the form of hydrochloric acid.

However, other buffer systems, e.g. B. a phosphate buffer can be used, unless they lead to undesirable side reactions. Suitable buffer systems are known to the person skilled in the art.

According to an advantageous development of the diagnostic device, the substance mixture additionally contains a compound which acts as a hydrogen or electron carrier. This compound catalyzes the reduction e.g. a tetrazolium salt to the corresponding colored formazan. For this purpose, the hydrogen or electron carrier must have a redox potential which is between the redox potential of the redox system used according to the invention (e.g. the NAD / NADH system) and the redox potential of the redox dye (e.g. the Tetrazolium salt).

Specific examples of such compounds are phenazine methosulfate (PMS), diaphorases, Meldola blue and menadione. Other corresponding compounds are known to the person skilled in the art.

The following formula shows an example of the reactions taking place when the diagnostic device is used if the NAD / NADH redox system, PMS are used as electron carriers and the tetrazolium salt NBT:

Carriers in the form of tampons made of cellulose fibers have proven particularly useful for diagnostic devices intended for the detection of carcinomas in the female genital area. The tampons are inserted into the vagina when they are used, thus allowing the mixture of substances on the tampon to react with the vaginal secretions. However, it is also possible to carry out the test extracorporeally, for example by N. Contact the vaginal secretion that has been removed outside the body with the impregnated tampon.

In principle, however, other carrier forms, such as rods or foils, can also be used.

The material of the carrier can have a fibrous, granular or other type of structure. In any case, the support has polar groups.

The invention also relates to the use of the diagnostic device for the early detection of pathological changes, in particular malignant diseases, in humans, animals and plants.

A preferred use of the diagnostic device is its use for the detection of a pathologically increased concentration of dehydrogenases in human, animal or vegetable extracellular liquids, the substance mixture on the carrier being reacted with the liquid to be examined, with the proviso that the reaction of the substance mixture with of the liquid to be examined is carried out for a maximum of 10 minutes. Optimal results are obtained if this time is 5 to 7 minutes.

This applies in particular to the case in which the carrier of the diagnostic device is a tampon which is inserted into the vagina of a woman to be examined for diagnosis. The tampon's lying time in the vagina then corresponds to the times mentioned above. If the lying time is more than 10 minutes, a color reaction on the tampon can occur in individual cases, even if there is no pathological change in the examined woman. It is therefore essential that the lay time is not chosen too long.

The optimal lay time of 5 to 7 minutes within the scope of the invention is considerably shorter than the preferred lay time of 15 to 30 minutes according to DE-PS 2 443 741. This is particularly important for the practicality.

In addition, it has proven particularly expedient if the patient walks around while the tampon is lying down in order to bring about a more intensive contact between the tampon and the vaginal skin in the vagina.

It has also been found that liquids that are to be checked for a pathologically increased dehydrogenase concentration can have very different pH values. For example, in women with pathological changes in the area of the genital organs, pH values of 3.9 to over 7 were measured for the vaginal secretions. Surprisingly, the diagnostic device according to the invention can be easily adjusted to such reaction conditions without losing diagnostic accuracy. For example, in the case of a liquid to be examined with a pH of 5.0 or higher, this value can be adjusted by means of a buffer system in the mixture of substances contained in the diagnostic device, which is set to a lower pH, for the implementation during the diagnosis in the be lowered as desired.

The following example explains the invention.

example

A series examination in various clinics checked the accuracy of the diagnostic device with a tampon as a carrier on 486 women with diagnosed cancer in the area of the genital organs. Table I below shows the distribution of the diagnostic groups across the various age groups in absolute numbers and percentages. The "OA" column (= without information) refers to the cases in which the examining clinic did not provide any information on this age distribution.

Explanations for individual diagnostic groups:

Collum carcinoma: the diagnostic terms collum carcinoma, cervical carcinoma and portiocarcinoma are summarized here.

Carcinomas of other locations and sarcomas:

Urethral carcinoma, breast sarcoma, malignant teratoma, uterine sarcoma, lymphosarcoma, fibrosarcoma, psammoma, malignant melanoma, gastric carcinoma, sigmoid carcinoma, rectal carcinoma, bladder carcinoma, lung carcinoma. The neoplasms were mainly included in the study because they had invasively or metastasized the genital apparatus.

Benign tumors:

Ovarian fibroma, ovarian, parovarial cyst, uterine myomatosus, corpus polyp, abdominal cyst, leiomyoblastoma.

Inflammation:

Salpingitis, tubo-ovarian abscess, cervicitis, colpitis, vaginitis, vulvitis, chronic. Pyelonephritis.

Diagnostic residual group:

Lower abdominal hematoma, menopausal bleeding, without diagnosis, without pathological findings.

The vaginal pH values of the patients were also measured as part of this investigation. The results are summarized in Table II below:

From the table it can be seen that the investigation covered a very wide pH range of the vaginal secretions.

Table III below shows the tampon lay times in minutes. This is to be understood as the time during which a diagnostic tampon remains in the vagina.

Tables IV, V, VI, VII, VIII, IX, X below summarize the test results for the accuracy of the diagnostic agent in the various diseases.

Explanation:

Table XI below summarizes the test results found in carcinomas.

It can be seen from this that the diagnostic device has a previously unattainable high accuracy and is suitable for differential diagnosis. The statistical evaluation of the test results showed that in all cases in which the presence of carcinomas (stage I to IV) in the genital area was ensured by other examination methods (292 cases), the diagnostic device was only 9.6% false-negative Diagnoses.

Claims (8)

1. Diagnostic device for the detection of an increased concentration of dehydrogenases and/or oxidases in the fluids of humans, animals or plants, which consists of a carrier which bonds a redox dyestuff, and a substance mixture, adjusted to a pH value in the acid range, of the substrate corresponding to the particular dehydrogenase, a hydrogen donor compound and at least one redox dyestuff, characterised in that use is made of a carrier which has polar groups and is formed of cellulose fibres, and the substance mixture for detecting exclusively pathologically increased concentrations of the particular dehydrogenase and/or oxidase is adjusted to a pH value of below 5.0.

2. Diagnostic device according to Claim 1, characterised in that the pH value is adjusted to at least 3.0.

3. Diagnostic device according to Claim 1 or 2, characterised in that the substance mixture contains a buffer system for establishing and/or maintaining the pH value.

4. Diagnostic device according to Claim 3, characterised in that the buffer system in the form of triethanolamine hydrochloride or in the form of a non-equivalent mixture of triethanolamine and hydrogen chloride.

5. Diagnostic device according to one of the preceding claims, characterised in that the substance mixture additionally contains a compound which acts as a hydrogen donor or electron donor.

6. Diagnostic device according to one of the preceding claims, characterised in that the carrier consists of a tampon containing cellulose fibres.

7. Use of the diagnostic device according to one of Claims 1 to 6 for itracorporal or extracorporal detection of a pathologically increased concentration of dehydrogenases in extracellular human, animal or vegetable fluids, the substance mixture on the carrier being reacted with the fluid to be investigated, characterised in that the reaction of the substance mixture with the fluid to be investigated is carried out for at most 10 minutes.

8. Use of the diagnostic device according to one of Claims 1 to 6, characterised in that the reaction is carried out for 5 to 7 minutes.